Integrase (IN) is encoded by the Pol gene from the HIV provirus and can be efficiently expressed as an active recombinant protein. Our laboratory has pioneered the integrase inhibitors research field (PNAS 1993), discovered several families of lead inhibitors (Nature Rev Drug Discovery 2005; Current Topics in Medicinal Chemistry 2009; Viruses 2010) and patented compounds for therapeutic development. Our current studies are focused on the discovery of novel chemotype integrase inhibitors to overcome resistance to raltegravir and target novel site of IN. We have discovered novel chemotypes derived from short Vpr peptides, which act as 3?-processing and strand transfer inhibitors. We have shown they could serve as antivirals by adding a poly-arginine tail to confer cellular uptake. A long-term goal is to build non-peptidic derivatives of those Vpr peptides. We have also published and patented novel synthetic chemotypes as IN strand transfer inhibitors (INSTIs) including phtalimide and quinolinonyl derivatives in collaborations with Dr. Terrence Burke, Laboratory of Medicinal Chemistry (CCR, NCI). We have reported novel INSTIs chemotypes with Dr. ZhengQuiang Wang and Dr. Robert Vince at the Center for Drug Design, University of Minnesota. And with Dr. Seth Cohen (Department of Chemistry and Biochemistry, USCD), we have probed the role of the metal-binding group as IN metalloenzyme inhibitors. To perform these experiments, we have developed a panel of recombinant IN proteins bearing the mutations observed in patients that develop resistance to raltegravir and elvitegravir. Using our set of raltegravir-resistant IN mutants, we have characterized the molecular pharmacology of elvitegravir, dolutegravir and MK-0536, comparing them to raltegravir. We have shown that both raltegravir and elvitegravir are highly selective for the strand transfer reaction, while being more than 100-fold less potent against the 3'-processing reaction, and almost inactive against the disintegration reaction mediated by integrase. The selective activity of raltegravir and elvitegravir against strand transfer (one of the 3 reactions mediated by integrase) demonstrates the very high specificity of the clinically developed strand transfer inhibitors. It is consistent with our pharmacological hypothesis (Nature Drug Discovery 2012) that the strand transfer inhibitors trap the IN-viral DNA complex by chelating the divalent metals in the enzyme catalytic site following 3'-processing of the viral DNA. We have characterized the biochemical enzymatic activities and drug sensitivities of the IN mutants that confer clinical drug resistance. We have expanded these studies to double-mutants in the integrase flexible loop that commonly arise in raltegravir-resistant patients. The working hypothesis is that the second mutation acts as gain of function to rescue the biochemical activity of IN after it had become defective by the presence of the first mutation. One of aims is to understand the molecular mechanisms of such complementation and the structural connections between the flexible loop, the viral and host DNAs, and the inhibitors. We found that the flexible loop double-mutant 140S-148H is cross-resistant to both raltegravir and elvitegravir but much less to dolutegravir and to some of our new derivatives. On the other hand, the 143Y mutant is primarily resistant to raltegravir and minimally resistant to elvitegravir and dolutegravir. These results provide a rationale for using elvitegravir in patients that develop resistance to raltegravir due to mutation 143Y (but not in the case of mutations 140S-148H). Our results support the value of elvitegravir, which is combined with 3 other drugs in the Quad pill (cobicistat to boost elvitegravir pharmacokinetics and two reverse transcriptase inhibitors, emtricitabine and tenofovir). The Gilead Quad pill received support of the FDA Advisory Committee in May 2012 for approval as once daily single tablet regimen for HIV. dolutegravir. We have also studied the molecular of action of dolutegravir, a lead compound from ViiV Healthcare, at the enzymatic and structural levels (Hare et al. 2011). Our results demonstrate that dolutegravir effectively inhibits a panel of HIV-1 IN variants resistant to raltegravir. To elucidate the structural basis for the increased potency of DTG against raltegravir-resistant INs, we determined crystal structures of wild type and mutant prototype foamy virus intasomes bound to the drug. This work was done in collaboration with Dr. Peter Cherepanov at the Imperial College in London(1). The overall IN binding mode of dolutegravir is strikingly similar to that of the tricyclic hydroxypyrrole MK-2048 (Merck & Co.). Both second-generation INSTIs occupy the same physical space within the IN active site and make contacts with the beta-4-alpha-2 loop of the catalytic core domain. Furthermore, the structures indicate that DTG displays considerable flexibility, particularly in the linker connecting the metal chelating core and the fluorophenyl group, allowing it to adjust to the structural changes in the active sites of the mutant integrases. The ability to structurally adapt to the structural changes associated with drug resistance appears to be a desirable characteristic that could be used in the development of new INSTIs. We also reported the biochemical and antiviral properties of MK-0536 (see Metifiot 2011). We synthesized this compound in collaboration with Dr. Terrence Burke (Chemical Biology Laboratory, CCR-NCI). MK-0536 was evaluated in the laboratory using our panel of recombinant mutant integrases while the antiviral assays where conducted in Dr. Steven Hughes laboratory (HIV Drug Resistance Program, CCR-NCI). We demonstrated that, like RAL, MK-0536 is highly potent against recombinant IN and viral replication. It is also effective against integrases that carry the three main raltegravir-resistance mutations (Y143R, N155H and to a lesser extent G140S-Q148H) and against the G118R mutant. Molecular models of integrases developed from the recent PFV structures, were generated in collaboration with Dr. Steven Hughes (Johnson et al. 2012). These models allow us to rationalize the differences between RAL and MK-0536 in the context of mutant integrases. We are continuing our long-term collaboration with Dr. Terrence Burke (Chemical Biology Laboratory, CCR-NCI). We recently developed a new pharmacophore model for second generation INSTIs. A patent has been filed and we recently published our most recent series of compounds (see Zhao et al. 2011). With Dr. Stephen Hughes, also at the NCI-Frederick (HIV Drug Resistance Program), we are testing the antiviral activity of those compounds and with Dr. Peter Cherepanov at the Imperial College in London we have recently obtained co-crystal structures. Our aim is to optimize our novel chemical series to obtain potent novel inhibitors active against clinically used INSTIs.